Many genetic diseases and defects are caused by only a minor mutation in a specific gene, such as a single-base mismatch (see, e.g., Cooper, D. N., et al., in “The Metabolic and Molecular Bases of Inherited Disease” (Scriver, C. R., et al., eds., (McGraw-Hill Inc., New York, Vol. 1, pp. 259-291 (1995)). Due to the high degree of sequence homology, therapeutic agents designed to inhibit the expression of a gene having a single (or multiple) point mutation almost inevitably affects the expression of the normal gene. Treating diseases that result from such genetic aberrations is problematic, particularly in proliferative diseases such as cancer, were expression of the non-mutated gene is essential for normal cellular function.
Double-stranded RNA molecules (dsRNA) have been shown to block gene expression in a highly conserved regulatory mechanism known as RNA interference (RNAi). Briefly, the RNAse III Dicer processes dsRNA into small interfering RNAs (siRNA) of approximately 22 nucleotides, which serve as guide sequences to induce target-specific mRNA cleavage by an RNA-induced silencing complex RISC (Hammond, S. M., et al., Nature (2000) 404:293-296). When administered to a cell or organism, exogenous dsRNA has been shown to direct the sequence-specific degradation of endogenous messenger RNA (mRNA) through RNAi. This phenomenon has been observed in a variety of organism, including mammals (see, e.g., WO 00/44895, Limmer; and DE 101 00 586 C1, Kruetzer et al.).
While completely complementary dsRNA are robust inhibitors of expression, Elbashir et al. have shown that dsRNA having a three-nucleotide mismatch with the target gene are very poor mediators of RNA interference (Elbashir, S. M., et al., Nature (2001) 411:494-498). On the other hand, Holen et al. have shown that dsRNA having one or two nucleotide mismatches can induce RNA interference, thereby inhibiting the expression of the target gene (Holen, T., et al., Nucl. Acid Res. (2002) 1757-1766). Thus, there appears to be no clear demarcation in activity upon which to design a dsRNA-based therapeutic agent for treating a disease resulting from a minor genetic aberration. Such an agent would likely produce serious side effects, due to the potential cross-reactivity between the mutant gene and its normal cellular counterpart gene.
While dsRNA can effectively silence a specific target gene, there is currently no available means for selectively inhibiting the expression of a gene comprising a point mutation without also inhibiting the expression of the normal, non-mutated gene. Thus, there remains a need for an agent that can selectively and efficiently silence a mutant gene without also affecting its wild-type counterpart. Compositions comprising such agents would be useful for treating genetic diseases and disorders caused by the expression of a gene having a minor mutation, such as a single or multiple-base mismatch.